SHORT REPORT

The GET pathway can increase the risk of mitochondrial outermembrane proteins to be mistargeted to the ERDaniela G. Vitali1, Monika Sinzel1, Elianne P. Bulthuis1,*, Antonia Kolb1, Susanne Zabel1,Dietmar G. Mehlhorn2, Bruna Figueiredo Costa3,‡, Ákos Farkas4, Anne Clancy4, Maya Schuldiner5,Christopher Grefen2, Blanche Schwappach4, Nica Borgese3 and Doron Rapaport1,§

ABSTRACTTail-anchored (TA) proteins are anchored to their correspondingmembrane via a single transmembrane segment (TMS) at their C-terminus. In yeast, the targeting of TA proteins to the endoplasmicreticulum (ER) can be mediated by the guided entry of TA proteins(GET) pathway, whereas it is not yet clear how mitochondrial TAproteins are targeted to their destination. It has been widely observedthat some mitochondrial outer membrane (MOM) proteins aremistargeted to the ER when overexpressed or when their targetingsignal is masked. However, the mechanism of this erroneous sortingis currently unknown. In this study, we demonstrate the involvement oftheGETmachinery in themistargeting of suboptimalMOMproteins tothe ER. These findings suggest that the GET machinery can, inprinciple, recognize and guide mitochondrial and non-canonical TAproteins. Hence, under normal conditions, an active mitochondrialtargeting pathway must exist that dominates the kinetic competitionagainst other pathways.

KEY WORDS: ER, GET, Mitochondria, Outer membrane,Protein sorting, Tail-anchor

INTRODUCTIONEukaryotic cells face the challenge of directing newly synthesizedmembrane proteins to the right compartment because theirmistargeting not only leads to their absence in the target organellebut also burdens the cytosol with aggregates of such proteins. Twomain destinations for such proteins are mitochondria and theendoplasmic reticulum (ER). The mechanisms for targeting eachmembrane protein to its correct membrane depend on the proteintopology and the targeting signals it contains.Hundreds of eukaryotic membrane proteins have a single

α-helical transmembrane segment (TMS) at their C-terminus(Kalbfleisch et al., 2007). The import of these proteins to the ERcan be mediated by the guided entry of tail-anchored (TA) proteins(GET) pathway (Schuldiner et al., 2008). The recognition happens

immediately after the release of the protein from the ribosome by thepre-targeting complex, which comprises Sgt2, Get4 and Get5. Sgt2binds the TMS and discriminates between mitochondrial and ERTA proteins (Wang et al., 2010). Sgt2 then hands over the substrateto the Get4−Get5 complex that, in turn, recruits Get3, a cytosolicchaperone. Get3 shuttles TA proteins to the ER membrane, whereGet1 and Get2 form a receptor complex that recognizes the Get3-TAprotein complex and facilitates the release of the TA proteins(Schuldiner et al., 2008). It appears that the Get1-Get2 receptor canmediate the membrane insertion of some TA proteins (Wang et al.,2011), however, other TA proteins with a moderately hydrophobicTMS, as e.g. cytochrome b5 and the protein tyrosine phosphatasePTP1B, can spontaneously insert into the lipid bilayer(Brambillasca et al., 2005; Colombo et al., 2009). Recently, anadditional ER membrane protein targeting pathway was identified,which can compensate the absence of either the signal recognitionparticle (SRP) or of the GET machinery and was named SRP-independent targeting (SND) pathway (Aviram et al., 2016;Hassdenteufel et al., 2017).

TA proteins are also targeted to the mitochondrial outermembrane (MOM), but none of the known mitochondrial importmachineries are required for their insertion (Kemper et al., 2008;Dukanovic and Rapaport, 2011). It has been proposed that thedifference in the lipid distribution (mainly of ergosterol) betweenER and mitochondria plays a role in assuring specificity in targetingto mitochondria (Krumpe et al., 2012). Compared to ER-localizedTA proteins, mitochondrial TA proteins generally have a moderatelyhydrophobic TMS flanked by positively charged residues. Despitethese differences, the overall similarity of targeting signals betweenER and mitochondrial destined TA proteins causes theirmistargeting to the wrong organelles on different occasions.However, the mechanism by which mistargeting occurs is, so far,unresolved.

In this work, we used Saccharomyces cerevisiae to identifyMOM proteins that are mislocalized to the ER because either theirtargeting sequence is masked or the membrane import machinery issaturated. We further demonstrate that their mistargeting to the ERmembrane depends on the GET machinery, suggesting that undernormal circumstances a mitochondrial targeting pathwaycounterbalances GET substrate capture.

RESULTS AND DISCUSSIONGET-dependent mislocalization of cytochrome b5-RRThe mammalian TA protein cytochrome b5 has two isoforms; one(b5-ER) is located in the ER and the other (b5-OM) in the MOM(D’Arrigo et al., 1993). The ER isoform has a predominantlynegatively charged C-terminus while the mitochondrial isoform ismostly positively charged. Replacement of the C-terminal segmentof b5-ER with two arginine residues – yielding substitution mutantReceived 4 October 2017; Accepted 10 April 2018

1Interfaculty Institute of Biochemistry, University of Tubingen, 72076 Tubingen,Germany. 2Centre for Plant Molecular Biology, Developmental Genetics, Universityof Tubingen, Tubingen 72076, Germany. 3Consiglio Nazionale delle RicercheInstitute of Neuroscience, Milan 20100, Italy. 4Department of Molecular Biology,Universitatsmedizin Gottingen, Gottingen 37073, Germany. 5Department ofMolecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.*Present address: Department of Biochemistry, Radboud University MedicalCentre, 6500HB Nijmegen, The Netherlands. ‡Present address: Telomeres andCancer Laboratory, Champalimaud Centre for the Unknown, 1400-038 Lisbon,Portugal.

§Author for correspondence (doron.rapaport@uni-tuebingen.de)

D.R., 0000-0003-3136-1207

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b5-RR – leads to re-direction of the protein to mitochondria(Borgese et al., 2001) (Fig. 1A).To understand better the distribution of the two isoforms between

both organelles, we expressed rabbit b5-ER and its b5-RR variant inyeast cells, and analysed their localization by subcellularfractionation. As expected, we found the vast majority of the ERform in the ER (microsomal) fraction of yeast cells and onlymarginal amounts in their mitochondria (Fig. 1B). Surprisingly,∼50% of the mitochondrial isoform was found in the ER fraction ofyeast cells (Fig. 1C). This is in sharp contrast to the situation inmammalian cells where the vast majority of b5-RR is found inmitochondria (Borgese et al., 2001). Thus, it seems that thosefeatures that assure correct targeting in mammalian cells do notfunction properly in yeast cells. Similar differences betweentargeting in mammalian cells compared with that in yeast wereobserved for PTP1B and Bcl2. In mammalian cells, both proteinslocalize to the ER andmitochondria but are found, once expressed inyeast cells, solely in the ER (Egan et al., 1999; Fueller et al., 2015).Furthermore, a substantial proportion of these b5-RR mistargeted

molecules migrated at a higher than expected molecular mass,suggesting that they had been modified (Fig. 1C). To characterizethe topology of the native and modified forms, we treated isolatedmicrosomes with proteinase K. This treatment resulted indisappearance of the native protein signal suggesting that itadopted a classical TA topology. In contrast, the modified form

was protease resistant, unless the membrane was solubilized withdetergent (Fig. 1D). This outcome raised the possibility that themodified form flipped its topology such that the N-terminus facesthe microsome lumen. Moreover, by using alkaline extraction bothnative and modified microsomal forms of b5-RR, as well as b5-RRlocalized in mitochondria, were found to be integrated intomembranes (Fig. 1E).

The inside-out topology of the modified b5-RR suggests that itsmodification might be glycosylation. Hence, we treated b5-RR-containing microsomes with either endoglycosidase H (EndoH) orpeptide:N-glycosidase (PNGase). Both enzymes caused thedisappearance of the modified form of b5-RR and of proteindisulfide-isomerase (Pdi1), which served as a control. Of note, theNetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/),which predicts N-glycosylation sites, suggested Asp residue 21 ofcytochrome b5 as a potential glycosylation site (Fig. 1A). Weconcluded that a considerable portion of b5-RR molecules wasmistargeted to the ER and some of those molecules had beeninserted in the opposite orientation, i.e. with the N-terminus in thelumen. These findings can be explained by recent reports suggestingthat the SRP and the Sec translocon are involved in the targeting ofsome TA proteins, including cytochrome b5, to the ER (Cassonet al., 2017; Hassdenteufel et al., 2017). Thus, it might be that theSec translocon mediates an integration of a sub-population of b5-RR into the ER membrane in the wrong topology.

Fig. 1. Cytochrome b5-RR is partiallymistargeted to ER in a GET-dependentmanner. (A) Schematic representation ofcytochrome b5 isoforms. Y represents a potentialglycosylation site. (B,C) Whole-cell lysate (WCL)and fractions corresponding to cytosol (cyt),microsomes (ER) and mitochondria (mito) fromcells expressing either b5-ER (B) or b5-RR (C)were analysed by SDS-PAGE andimmunoblotting. (D) Western blot showing ERfractions treated with proteinase K (PK) in theabsence or presence of Triton X-100 (TX).(E) Western blot showing ER and mitochondriafractions subjected to alkaline extraction. Pellet(P) and supernatant (S) fractions were analysedby SDS-PAGE and immunoblotting. (F) Westernblot showing the ER fraction incubated in thepresence (+) or absence (−) of either EndoH orPNGase. (G) Western blot showing WT, get1Δand get3Δ cells expressing b5-RR subjected tosubcellular fractionation and analysis as in (C).(H) Quantification of three independentexperiments as in G; enrichment of the lower formof b5-RR in ER fractions is depicted. Arrowheadsin C-G indicate the modified form of HA-b5-RR.

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Since ER TA proteins can be targeted to their destination by theGET machinery (Borgese and Fasana, 2011; Schuldiner et al.,2008), we wondered whether this system can participate in themissorting of b5-RR. To test this, we expressed b5-RR in cells thatlack the ER receptor Get1 or the cytosolic chaperone Get3. Weobserved that, in both deletion strains, a smaller proportion of b5-RR molecules localized to the ER, whereas higher amounts werefound in mitochondria (Fig. 1G,H). These findings suggest that theGET machinery deviates this substrate from its natural targetmembrane. Of note, we observed that ∼30-40% of b5-RRmolecules are localized to ER, even in the absence of functionalGET system. This partial dependence on the GET components is inline with the idea that multiple selection filters are used by the GETmachinery to assure correct targeting (Rao et al., 2016), and thatalternative pathways, involving SRP, hSnd2 and/or unassisted

membrane integration, exist for ER TA protein targeting in theabsence of GET (Casson et al., 2017; Hassdenteufel et al., 2017).

The GET machinery mediates mistargeting of Mcp3In S. cerevisiae the MOM protein Mcp3 follows a unique importpathway that involves the TOM and TIM23 complexes, as well asprocessing by the inner membrane peptidases 1 and 2 (Imp1/2)(Sinzel et al., 2016). Mcp3 contains a presequence-like segment inits N-terminal region, whereas the C-terminal half contains twoputative TMSs, one of them very close to the C-terminus (Fig. 2A).When Mcp3 was N-terminally labelled with GFP, we observedconsiderable mislocalization to the ER (Fig. 2B), potentially due tomasking of the presequence by the GFP moiety.

Of note, alkaline extraction confirmed that the GFP-taggedversion was integrated into the membranes of either mitochondria or

Fig. 2. The mistargeting of GFP-Mcp3 to ERrequires the GET machinery. (A) Schematicrepresentation of GFP-Mcp3. (B) Western blotshowing cells expressing GFP-Mcp3 subjected tosubcellular fractionation and analysis as describedfor Fig. 1B. (C) Western blot showing ER andmitochondrial fractions subjected to alkalineextraction as described for Fig. 1E. T, total; P, pellet;S, supernatant. (D) WT, get3Δ, get1Δget2Δ, andget1Δget2Δget3Δ cells expressing GFP-Mcp3 andeither the ER marker HDEL-dsRed or themitochondrial marker mt-dsRed were analysed byfluorescence microscopy and representative imagesare shown. Arrowheads indicate perinuclear ERlocalization. Scale bars: 5 µm. (E) Quantification ofthe intracellular localization of GFP-Mcp3 monitoredas in D. The figure shows the average±s.d. of threeindependent experiments with at least 100 cellseach. (F-H) Western blots showing WT, get3Δ (F),get1/2Δ (G), and get1/2/3Δ (H) cells expressingGFP-Mcp3 subjected to subcellular fractionation asdescribed for Fig. 1B. WCL, whole-cell lysate; cyt,cytosol fraction; ER, microsome fraction; mito,mitochondria fraction; WT, wild type.

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the ER (Fig. 2C). SinceMcp3 has a TMS at its C-terminal region, wewondered whether GET components are required for its missorting.To address this point, we introduced GFP-Mcp3 into strains deletedfor GET3 alone (get3Δ), double-deleted for GET1 and GET2 (get1/2Δ), or triple deleted for GET1, GET2 and GET3 (get1/2/3Δ).Fluorescence microscopy verified the predominant ER localizationof GFP-Mcp3 inWT cells. In sharp contrast, only negligible stainingof the ER and a typical tubular pattern of mitochondria was observedin cells lacking one, two or all of the GET components (Fig. 2D,E).To test our assumption that the N-terminal GFP interferes with

the function of the presequence of Mcp3, we constructed a Mcp3variant lacking its N-terminally presequence (Mcp3ΔN). Indeed,this construct behaved similarly to the GFP full-length Mcp3 andwas localized to ER structures. This location disappeared upondeletion of either GET1 or GET3 (Fig. S1). However, in contrast tothe full-length protein, the truncated variant, which lacks themitochondrial targeting signal, was spread in the absence of theGET machinery in the cytosol or appeared in punctate structures,representing probably aggregated molecules (Fig. S1).To support the fluorescence microscopy data, we performed

subcellular fractionation of WT cells and get mutant cellsexpressing GFP-Mcp3. In all get mutant strains, we observedmuch higher amounts of GFP-Mcp3 in the mitochondrial fraction ascompared to WT cells (Fig. 2F-H). Notably, the get mutant strainsappear to contain a minor population of GFP-Mcp3 in their ERfraction. This, again, might be due to alternative targeting pathwayssupporting this rerouting but could also be due to cross-contamination between the ER and mitochondrial fractions.Markedly, the overall higher amounts of GFP-Mcp3 in the getmutants raise the possibility that GFP-Mcp3 is unstable in WT cellsand undergoes degradation.In summary, masking the mitochondrial targeting information in

the N-terminal region with a GFP moiety probably slowed theassociation with mitochondria, thus providing the GET machinery achance to recognize the C-terminal TMS of Mcp3 as a potentialsubstrate. In the case of native Mcp3, the mitochondrial import ismost likely so fast that it does not provide the GETmachinery a timewindow to interfere with this process.

Overexpressed GFP-tagged Mim1 is partially targeted tothe ERThe yeast mitochondrial import protein 1 (Mim1) is a MOM proteinthat harbours a central membrane-spanning hydrophobic stretch(Ishikawa et al., 2004; Waizenegger et al., 2005) (Fig. 3A).Subcellular fractionation indicated that, upon overexpression, GFP-Mim1 is mistargeted to the ER (Fig. 3B). It has been suggested thatthe GET pathway can also recognize TMSs that are not strictly at theC-terminus (Aviram et al., 2016), so it remained possible that it caneven recognize proteins with a central TMS, like Mim1.To understand better the mechanism of mistargeting, we first

assayed whether the missorted overexpressed GFP-Mim1 ismembrane-embedded, and observed that GFP-Mim1 behaved as amembrane protein in both ER and mitochondria fractions (Fig. 3C,D).We next investigated whether the ER localization is dependenton GET proteins. Hence, we expressed GFP-Mim1 in get1Δor get3Δ cells and analysed the protein localization by fluorescencemicroscopy. Whereas in WT cells ∼20% of the cells had ERstaining, only a negligible proportion of the get mutant cellsdisplayed the GFP signal in the ER (Fig. 3E,F). We further checkedthe distribution of GFP-Mim1 inWT and getmutants by subcellularfractionation. Importantly, the amount of GFP-Mim1 in the ER was

significantly reduced in the get deletion strains (Fig. 3G-I). Thepresence of a residual ER population of the protein, despite deletionof GET components, suggests that the GET pathway is not the onlyroute for GFP-Mim1 targeting to the ER.

Next, we wondered if the mislocalization depends on thepresence of the GFP moiety and on its location. To test this, wefused GFP to the C-terminus of Mim1 and analysed the subcellulardistribution of the fusion protein. We observed the vast majority ofthe protein in the mitochondrial fraction, whereas only a minoritywas mistargeted to the ER (Fig. S2A). Similarly, overexpresseduntagged Mim1 was very partially mislocalized to the ER where itwas modified in WT, but not in get3Δ cells (Fig. S2B). Thismodification does not appear to be glycosylation (Fig. S2C), and itis not clear to us why we did not observe it in get3Δ cells. Of note,the GET machinery does not seem to contribute to the mistargetingof both Mim1 and Mim1-GFP (Fig. S2A,B). This finding is inagreement with the location of the TMS being positioned in themiddle of the protein (as in Mim1) or in its N-terminal region (as inMim1-GFP), rather than in the C-terminal region (as in GFP-Mim1).

Get3 interacts directly with Mcp3 and Mim1The results described above, suggest that the GET machinery isinvolved in mistargeting of mitochondrial proteins. To test whetherthis effect is a direct one, we expressed a His-tagged version of thesoluble component Get3 or of its ATP hydrolysis-deficient mutant(D57N) (Stefer et al., 2011), which fails to release substrate proteins,in E. coli cells. The purified proteins were incubated with rabbitreticulocyte lysate expressing HA-Mim1 or HA-Mcp3ΔN, orDHFR-HA as a control. Next, a pull-down with anti-HA beadswas performed and bound proteins were analysed. While we coulddetect only minor binding of native Get3 to HA-tagged proteins, thefraction of bound Get3 was much larger for the ATP hydrolysis-deficient mutant D57N (Fig. 4A,B). Of note, none of the Get3variants was bound to the control protein, DHFR. Thus, these resultsindicate that Get3 is able to bind in vitro to mitochondrial proteins.

To substantiate these findings by an in vivo approach, weemployed the cytosolic Split-Ubiquitin System (Asseck et al., 2018;Xing et al., 2016). To this end, we used Get3 as a bait, whereasMcp3ΔN or GFP-Mcp3ΔN were utilized as preys. Indeed, usingthese combinations, we observed growth of the yeast cells onstringent Met-containing growth medium, whereas the usage of thenegative control NubG as a prey did not result in growth under theseconditions (Fig. 4C). Hence, we conclude that Get3 is able tointeract in vivo with Mcp3.

ConclusionsOur study shows that, when allowed to, the GET pathway is ableto recognize newly synthesized mitochondrial proteins. However,this capacity becomes relevant only when the mitochondrial importis compromised. Under normal conditions, the high efficiencyand fast kinetics of the mitochondrial import apparatus do notprovide factors involved in ER-targeting routes with the option tosuccessfully compete for such interactions. This implies that correctintracellular targeting is dictated by a kinetic competition amongvarious potential pathways.

MATERIALS AND METHODSYeast strains and growth conditionsYeast strains used in the study were isogenic to Saccharomyces cerevisiaestrainW303α or BY4741. Standard genetic techniques were used for growthand manipulation of yeast strains.

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Yeast cells were grown in standard rich medium YP (2% [w/v] bactopeptone, 1% [w/v] yeast extract) or synthetic medium S (0.67% [w/v] bacto-yeast nitrogen base without amino acids) with either glucose (2% [w/v], D)or galactose (2% [w/v], Gal) as carbon source. Transformation of yeast cellswas performed by the lithium acetate method.

To delete the complete ORFs of GET1, GET2 or GET3, they werereplaced with KanMX4, CloNAT or Ble cassettes amplified with gene-specific primers. The deletions were confirmed by PCR. The GFP-tag at theN-terminus of theMCP3ORF was genomically inserted and encoded underthe SpNOP1 promoter. A GFP-moiety was inserted upstream of the MIM1ORF and the fusion protein was expressed under the control of the ADHpromoter. Table S1 includes a list of strains used in this study.

Recombinant DNA techniquesThe cDNAs of rabbit cytochrome b5 ER and its RR variant were amplifiedby PCR with primers containing EcoRI and HindIII restriction sites from

pGEM4-b5ER and pCDNA3-b5RR, respectively (Borgese et al., 2001).The obtained DNA fragments were inserted in-frame with an N-terminal3HA-tag that was cloned between EcoRI and NcoI sites, into the multi-copyyeast expression plasmid pYX223 (GAL promoter). To obtain pGEM4-yk-DHFR-3HA, the DHFR coding sequence was amplified from pGEM4-pSu9-DHFR with primers containing KpnI and BamHI restriction sites aswell as the yeast Kozak sequence, and inserted into the pGEM4 plasmid in-frame with a C-terminal 3HA-tag cloned into BamHI and SalI restrictionsites.

Plasmid pRS426-TPI-GFP-Mcp3ΔNwas obtained by PCR amplificationfrom genomic DNA, of the sequence coding for the 126 most C-terminalamino acids of Mcp3, with primers containing BamHI and HindIIIrestriction sites. The obtained DNA fragment was inserted in the pRS426-TPI vector in-frame with an N-terminal GFP cloned between two EcoRIsites. The MCP3ΔN coding sequence was subcloned, by using BamHI andHindIII restriction enzymes, from this plasmid into a pGEM4 vector

Fig. 3. GET proteins are involved in themislocalization of GFP-Mim1 to ER.(A) Schematic representation of GFP-Mim1. (B-D)Western blot showing cells expressing GFP-Mim1subjected to subcellular fractionation (B). (C,D)Western blots of ER (C) and mitochondrial (D)fractions subjected to alkaline extraction. (E) WT,get1Δ, and get3Δ cells expressing GFP-Mim1 wereanalysed by fluorescence microscopy as describedin the legend to Fig. 2D. Scale bars: 5 μm.(F) Quantification of the intracellular localization ofGFP-Mim1 monitored as in E and analysed asdescribed in the legend to Fig. 2E. **P≤0.001;***P≤0.0001. (G-H)Western blots ofWT, get3Δ (G),and get1Δ (H) cells expressing GFP-Mim1subjected to subcellular fractionation. (I) Threeindependent experimentsasshown inGandHwerequantified, and the enrichment of GFP-Mim1 in ERfractions are depicted. WCL, whole-cell lysate; cyt,cytosol fraction; ER, microsome fraction; mito,mitochondria fraction; WT, wild type.

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containing the yeast Kozak sequence and an N-terminal 3HA-tag betweenEcoRI and KpnI sites. The ORF coding for Mim1 was amplified by PCRfrom pRS426-TPI-MIM1 with primers containing restriction sites BamHIand HindIII, and fourMet residues at the C-terminus. The obtained fragmentwas inserted in-frame with an N-terminal 3HA-tag, which was clonedbetween the EcoRI and KpnI restriction sites, into a pGEM4 vectorcontaining the yeast Kozak sequence. To obtain the construct Mim1-GFP,the MIM1 ORF without a stop codon was PCR amplified with primerscontaining EcoRI and BamHI restriction sites. Then, the PCR product wastreated with both restriction enzymes and was inserted into the pRS426-TPIvector in-frame with a C-terminal GFP, which was inserted between KpnIand HindIII restriction sites. Similarly, theMIM1 ORF was inserted into thepYX223 vector using EcoRI and HindIII.

Biochemical methodsProtein samples for immunoblotting were analysed on 12.5% or 15% SDS-PAGE and subsequently transferred onto nitrocellulose membranes bysemi-dry western blotting. Proteins were detected by incubating themembranes, first with primary antibodies and then with horseradishperoxidase-conjugates of goat anti-rabbit or goat anti-rat secondaryantibodies. Band intensities were quantified using the AIDA software(Elysia-raytest, Straubenhardt, Germany). Enrichment in the ER fractionwas calculated by dividing the signal for the protein of interest in the ERfraction by that in the whole-cell lysate. This value was then divided by thesame ratio calculated for the marker ER protein, Erv2 or Sec61 (protein X inER/protein X in WCL)/(Erv2 or Sec61 in ER/Erv2 or Sec61 in WCL).

Subcellular fractionationwas performed as described before (Walther et al.,2009). Isolationofmitochondria fromyeast cellswasperformedbydifferentialcentrifugation, as previously described (Daum et al., 1982). To obtain highlypuremitochondria, isolated organelleswere layeredon top of aPercoll gradientand isolated according to a published procedure (Graham, 2001).

For protease protection assay, 50 µg of microsomes were resuspended in100 µl of SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM MOPS pH

7.2). As a control, microsomes were treated with 1% Triton X-100 in SEMbuffer and incubated on ice for 30 min. The samples were supplementedwith proteinase K (50 µg/ml) and incubated on ice for 30 min. Theproteolytic reaction was stopped with 5 mM phenylmethylsulfonyl fluoride(PMSF). The samples were precipitated with trichloroacetic acid (TCA) andresuspended in 40 µl of 2× Laemmli buffer, heated for 10 min at 50°C, andanalysed by SDS-PAGE and immunoblotting.

To analyse the membrane topology of proteins, alkaline extraction wasperformed. Mitochondria or ER fractions (50 µg) were resuspended in100 µl of buffer containing 10 mM HEPES-KOH pH 11.5 with 100 mMNa2CO3, and incubated on ice for 30 min. The membrane fraction waspelleted by centrifugation (76,000 g, at 2°C for 30 min) and the supernatantfraction was precipitated with TCA. Both fractions were resuspended in40 µl of 2× Laemmli buffer, heated for 10 min at 50°C or 95°C, andanalysed by SDS-PAGE and immunoblotting.

The following proteins were used as marker proteins in western blotsshown in Figs 1-4: Bmh1, a cytosolic protein; Erv2, an ER membraneprotein exposed to the ER lumen; Fis1, a mitochondrial membrane protein;Hep1, a soluble mitochondrial protein; Om14, a mitochondrial membraneprotein; Pdi1, a soluble glycosylated ER protein; Tom70, a mitochondrialmembrane protein; Sec61, an ER membrane protein; Tob55, amitochondrial membrane protein; Tom40, a MOM protein. Table S1includes a list of the antibodies used in this study.

In vitro interactions of recombinant Get3Plasmids encoding His-tagged versions of Get3 and of its ATP hydrolysis-deficient mutant (D57N) were a kind gift from Irmgard Sinning. Proteinswere expressed in E. coli cells and purified as described previously (Steferet al., 2011). 3HA-Mim1, 3HA-Mcp3ΔN or DHFR-3HAwere translated invitro in rabbit reticulocyte lysate in the presence of 10 mMDTT and 5 µM ofrecombinant Get3-6His or Get3D57N-6His. After translation, the lysate wasdiluted with KHM buffer (110 mM KAc, 20 mM HEPES-KOH pH 7.4,2 mMMgCl2) supplemented with 50 mM ATP. Then, the lysate was added

Fig. 4. Get3 physically interacts with Mcp3 and Mim1.(A,B) The indicated radiolabelled HA-tagged proteins wereincubated with buffer only (-) or with His-tagged versions ofeither native Get3 (WT) or the D57N variant. The mixtureswere pulled-down with anti-HA beads. Samples from the inputand the eluates were analysed by SDS-PAGE andimmunoblotting. (C) The cytoSUS was used to monitorinteraction of Get3 (used as bait) with Mcp3ΔN (used as prey)with or without GFP-tag together with controls (NubG,negative; NubI, positive). Diploid yeast cells were dropped atOD600 of 1.0, 0.1 and 0.01 on complete supplement mixture(CSM) medium to verify mating and on CSM with either 50 or500 µM methionine to test for the specificity of interaction.

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to magnetic anti-HA beads (10 µl) that had been equilibrated with KHMbuffer for 30 min at 4°C, and incubated with them for 2 h at 4°C. The beadswere washed four times with KHM buffer and bound proteins were eluted ateither 95°C or 50°C for 10 min with 100 µl of 2× Laemmli buffer lackingβ-mercaptoethanol but supplemented with 5% H2O2. Samples wereanalysed by SDS-PAGE and immunoblotting.

Glycosylation assayTo test for glycosylation of proteins, 50 µg of the ER fraction wasresuspended in 10 µl glycoprotein denaturing buffer (0.5% SDS, 40 mMDTT) and incubated for 10 min at 95°C. Then, the samples weresupplemented with 500 units of either endoglycosidase H (EndoH) orpeptide:N-glycosidase F (PNGase) (New England BioLabs) in therespective buffer (according to the manufacturer’s instructions) andincubated for 1 h at 37°C. At the end of the incubation period, thesamples were precipitated with TCA, resuspended in 40 µl of 2× Laemmlibuffer, heated for 10 min at either 50°C or 95°C, and analysed by SDS-PAGE and immunoblotting.

The yeast cytosolic split-ubiquitin systemThe yeast cytosolic split-ubiquitin system (cytoSUS) was used to detectphysical interaction. The bait protein Get3 was expressed from the Met25promoter, N-terminally fused to the transmembrane domain of OST4p(mOST4) to ensure membrane anchoring and C-terminally tagged with theC-terminal ubiquitin moiety (Cub) followed by the chimeric ProteinA-LexA-VP16 (PLV) transcription activator (Xing et al., 2016). The baitfusion was transformed in the S. cerevisiae strain THY.AP4. N-terminallyNubG-2×HA-tagged prey proteins GFP-Mcp3ΔN and Mcp3ΔN, as well asthe control peptides NubG (as a positive control) and NubI (wild-type Nub,as a positive control) were transformed in the S. cerevisiae strain THY.AP5.After mating, diploids were selected. Interaction analysis was performed byspotting serial dilutions of diploid yeast on interaction-selective completesupplement mixture (CSM) medium lacking adenine and histidine butcontaining increasing concentrations of methionine (50–500 µM) todecrease bait expression. Protein expression was verified by westernblotting utilizing anti-VP16 antibody (rabbit, GeneTex) for bait and anti-HAperoxidase-conjugated (Roche) antibody for prey fusions as describedpreviously (Asseck et al., 2018; Xing et al., 2016).

Fluorescence microscopyFluorescence images were acquired using a spinning disk microscope (ZeissAxio Examiner Z1) equipped with a CSU-X1 real-time confocal system(Visitron), VS-Laser system and SPOT Flex CCD camera (VisitronSystems). Images were analysed with VisiView software (Visitron).Microscopy images of strains expressing GFP-Mim1 were acquired withan Axioskop 20 fluorescence microscope equipped with an Axiocam MRmcamera using the 43 Cy3 filter set and the AxioVision software (Carl Zeiss).

AcknowledgementsWe thank E. Kracker for excellent technical assistance and Irmgard Sinning,Biochemistry Center (BZH), Heidelberg, Germany, for plasmids.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: D.G.V., C.G., B.S., N.B., D.R.; Methodology: D.G.V., E.P.B.,M. Sinzel, A.K., S.Z., D.G.M., A.C., A.F., B.F.C., C.G., D.R.; Formal analysis: D.R.;Investigation: D.G.V., E.P.B., M. Sinzel, A.K., S.Z., A.C., B.F.C., C.G.; Resources:A.C., A.F., M. Schuldiner, B.S., N.B., D.R.; Data curation: E.P.B., M. Sinzel, A.K.,S.Z., D.G.M., C.G.; Writing - original draft: D.G.V., M. Schuldiner, B.S., N.B., D.R.;Writing - review & editing: C.G.; Supervision: B.S., N.B., D.R.

FundingThis work was supported by the Deutsche Forschungsgemeinschaft(Sonderforschungsbereich 1190-TP04 to B.S.; RA 1028/7-1 to D.R.; Deutsch-Israelische Projektkooperation to D.R. andM. Schuldiner; andGR 4251/2-1 to C.G.),and the ITN TAMPting network to D.G.V., B.F.C., B.S., N.B. and D.R. [funded by the

People Programme (Marie Curie Actions) of the European Union’s SeventhFramework Programme FP7/2007-2013/ under REA grant agreement no 607072].

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.211110.supplemental

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